This invention relates to the field of genetically engineered, redirected immune cells and to the field of cellular immunotherapy of CE7 malignancies.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice are incorporated by reference.
Neuroblastoma, a neoplasm arising from sympathetic ganglion cells, is the most common extracranial solid tumor of childhood and is third in incidence among pediatric malignancies after the leukemia-lymphoma syndromes and central nervous system tumors [1–2]. Approximately 550 new cases occur annually in the United States; seventy-nine per cent of children are diagnosed prior to their fifth birthday. Stage of disease at diagnosis, patient's age at diagnosis, and characteristics of tumor cells such as histologic appearance and NMYC gene amplification are important prognostic factors which can be utilized to categorize patients into low, intermediate and high risk for poor outcome [3,4]. While survival for low and intermediate risk neuroblastoma is excellent, the prognosis for high-risk neuroblastoma remains dismal. Despite improved tumor response rates following intensive multi-modality therapy the median response duration of high risk tumors is less than 1 year and less than 40% of patients with high risk neuroblastoma survive more than 2 years [3–6]. To date there are no treatment modalities with proven efficacy for salvaging children with recurrent/refractory disseminated neuroblastoma.
Current treatment strategies for neuroblastoma are tailored to risk-stratified algorithms based on the International Neuroblastoma Staging System (INSS) [6–9]. The primary risk factors accounted for are age at diagnosis, stage of disease, tumor histology, NYMC copy number, and DNA index. High-risk disease includes those children greater than twelve months of age with tumor dissemination (Stage IV) as well as Stage 3 and 4 disease with unfavorable histology or NMYC amplification, regardless of age [10–17]. High-risk disease accounts for more than half of newly diagnosed cases of neuroblastoma and approximately three-quarters of cases diagnosed in children greater than 12 months of age. A multi-modality maximally intensive approach to treatment of high-risk neuroblastoma has evolved that includes aggressive induction chemotherapy, surgery, radiation therapy, autologous stem cell transplantation, and post-transplant biologic therapy with cis-retinoic acid. Two successive Children's Cancer Group trials (CCG-321, CCG-3891) have demonstrated improved survival for those patients receiving myelo-ablative therapy compared to those patients receiving conventional chemotherapy [18]. Aggressive local therapy including complete surgical resection of the primary tumor and local radiation appears to decrease the incidence of primary site recurrence. Unfortunately, more than 50% of all patients and 75% of patients failing to achieve a complete remission with 1st line conventional therapy continue to develop recurrent disease. Relapses typically occur in a disseminated fashion within the first 24-months after completing front-line therapy. Responses to salvage chemotherapy are limited and generally are not durable with only 8% of patients surviving greater than 3 years from time of recurrences [19–23].
Disease relapse for many children with neuroblastoma frequently occurs following the induction of a clinical complete response with standard treatment modalities, demonstrating that the persistence of minimal residual disease is a major obstacle for curative therapy. However, patients heavily treated with chemotherapy, radiation, surgery, and autologous transplantation have a limited capacity to tolerate additional cytotoxic therapeutic modalities to target minimal residual disease. The potential of targeting a limited tumor burden with immune-based approaches is attractive both, because of the opportunity to invoke immunologic effector mechanisms to which chemotherapy/radiation-resistant tumor cells are susceptible, as well as the limited toxicity theoretically possible with tumor-specific immunologic effector mechanisms.
Passive immunotherapy for neuroblastoma utilizing murine monoclonal and murine/human chimeric monoclonal antibodies have focused primarily on targeting the GD2 disialoganglioside present at high density of human NB [24–26]. Cheung et al. have investigated in clinical trials the GD2-specific monoclonal antibody 3F8 and have reported on the safety and, more recently, the anti-tumor activity of antibody therapy in the setting of minimal residual disease [27]. The long-term outcome of patients treated with 3F8 awaits delineation, however, limitations in its use have been observed early after treatment due to the development of neutralizing HAMA responses in approximately a third of patients [27]. Additionally, failure of antibody therapy to target MRD in the CNS due to poor penetration of immunoglobulin across the blood-brain-barrier was manifested by an unusually high incidence of isolated CNS relapses in antibody treated patients.
The cloning and production of recombinant cytokines have facilitated their introduction into clinical trials designed to activate and expand immunologic effector cells in vivo. Various cytokines either along or in combination have been evaluated in preclinical neuroblastoma animal models [28–30]. Interleukin-2 administration following transplantation has been most extensively studied as a strategy to activate NK cells and induce LAK cells. IL-2 therapy for patients with recurrent metastatic neuroblastoma failed to provide anti-tumor activity in 15 children treated [31,32]. These studies to date have revealed a significant incidence of severe toxicities associated with high-dose IL-2 administration without a clear impact on decreasing disease relapse. The prolonged use of low-dose IL-2, although not having demonstrable anti-neuroblastoma activity, can be administered to heavily pre-treated children without severe toxicity [33]. Pession et al. have reported on the administration of 212 courses of low dose IL-2 to 17 children with neuroblastoma following stem cell transplantation [33]. These maintenance courses were delivered bimonthly over five days/course at IL-2 doses of 2×106 U/m2/day escalating to 4×106 U/m2/day. No life-threatening toxicities were encountered. Fever controlled by acetaminophen and transient rash were the most common side effects of therapy. Consequently, current Phase I studies are evaluating the combination of cytokines that activate effector cells operative in antibody dependent cellular cytotoxicity (IL-2 and GM-CSF) in combination with anti-GD2 antibody therapy [34,35]. Frost et al. have investigated the use of monoclonal anti-GD2 antibody, 14.G2a plus IL-2 in 31 children with refractory neuroblastoma. Dose limiting toxicities included generalized pain and fever without documented infection. Tumor progression was noted in 63% of patients. Of note, 30% of patients with evaluable bone marrow disease had a significant decrease in quantity of tumor cells detected by immunohistochemical analysis [35]. The engineering of antibody-cytokine fusion molecules appears to potentiate the anti-tumor activity of either molecule administered separately in animal models, these fusion proteins are currently under investigation in clinical trials [36,37].
Induction or augmentation of a cellular immune response against neuroblastoma is an attractive strategy for eliminating resistant tumor cells. The availability of recombinant interleukin-2 (IL-2) and the demonstration that lymphocytes cultured in high concentrations of lymphokines acquire the ability to lyse, in a non-MHC-restricted fashion, a variety of tumor types, led to trials attempting to target neuroblastoma with the adoptive transfer of autologous ex-vivo expanded LAK cells [38]. Up to 1011 LAK cells have been administered in a single intravenous infusion to cancer patients without dose-limiting side effects, demonstrating the safety of adoptive therapy with large numbers of in vitro activated autologous lymphocytes. The toxicity that has been observed in these trials was attributed solely to the systemic effects of high-dose IL-2 that is required to support LAK cells in vivo [39]. LAK cell therapy in children with neuroblastoma has met with significant toxicities without obvious clinical benefit [31].
Animal models as well as a small but growing number of human tumor systems have demonstrated that anti-tumor cellular immune responses can be invoked or amplified by vaccination with tumor cells genetically modified to have enhanced immunogenicity. Transgenes that are being evaluated for neuroblastoma tumor cell vaccines include allogeneic HLA class II molecules, the co-stimulatory ligand B7-1, and pro-inflammatory cytokines [40–46]. Recently Bowman et al. published their results of a pilot study in which ten children with relapsed advanced stage neuroblastoma were treated with autologous tumor cells genetically modified to secrete IL-2 [47]. Of note, five patients had objective systemic anti-tumor responses correlating with the development of in vitro detected anti-tumor cellular cytotoxicity. These studies provide a glimpse at the potential of cellular immunotherapy for neuroblastoma but underscore the variability of inducing clinically relevant anti-tumor responses with vaccines and the technical difficulties in generating autologous genetically manipulated tumor cell lines for this application.
Antigen-specific T cells are immunologic effector cells that confer protection from lethal tumor challenge in animal models [48]. Adoptive transfer of tumor-specific T cell clones into tumor bearing hosts can eradicate established disseminated tumors. Enomoto et al. have demonstrated in a murine model system employing a poorly immunogenic syngeneic neuroblastoma, the capacity of adoptively transferred tumor-reactive cytotoxic T lymphocytes (CTL) to eradicate disseminated nueroblastoma [49]. This provocative model system, in light of the responses seen clinically to IL-2 producing tumor vaccine administration, suggest that adoptive therapy with neuroblastoma-specific T cells may have significant clinical utility provided these T cells can be reliably isolated from this patient population.
An ideal cell-surface epitope for targeting with antigen-specific T cells would be expressed solely on tumor cells in a homogeneous fashion and on all tumors within a population of patients with the same diagnosis. Modulation and/or shedding of the target molecule from the tumor cell membrane may also impact on the utility of a particular target epitope for re-directed T cell recognition. To date few “ideal” tumor-specific epitopes have been defined and secondary epitopes have been targeted based on either lack of expression on critical normal tissues or relative over-expression on tumors. Anti-GD2 antibodies have been most extensively utilized in antigen-specific immunotherapy for neuroblastoma. GD2, however, is expressed on peripheral nerves as well as brain grey matter. T cells, unlike antibody, can extensively access the CNS blood-brain barrier making GD2 re-directed adoptive T cell therapy subject to potentially severe neurologic toxicities [50].
Several groups have generated murine monoclonal antibodies reactive with human neuroblastoma by immunization of mice with human NB tumor cell lines. Blaser et al. have published on the generation of the CE7 monoclonal antibody (γI/κ) raised by immunizing mice with the IMR-32 human neuroblastoma cell line. CE7 uniformly binds to human neuroblastoma cell lines and primary tumors [51–53]. This high affinity IgGI monoclonal antibody (Ka=10−11) precipitates a 190-kDA plasma membrane-associated glycoprotein [54]. Tumor cells express in excess of 40,000 binding epitopes for CE7 and the target molecule does not shed from the cell surface. Biodistribution studies in nude mice revealed that up to 32% of injected dose/g tissue of iodinated antibody accumulates in tumor explants with low blood and organ uptake [55,56]. Importantly, this antibody in immunohistochemistry screening of normal tissues failed to bind to all non-neuroectodermal tissues as well as brain [51]. Carrel et al. have generated a mouse/human chimeric antibody and are pursuing preclinical studies for development of CE7-targeted radioimmunotherapy for neuroblastoma [56]. As with many monoclonal antibodies raised against tumor cell lines, the molecular identity of the target epitope of CE7 awaits delineation.
The safety of adoptively transferring antigen-specific CTL clones in humans was originally examined in bone marrow transplant patients who received donor-derived CMV-specific T cells [57,80]. Studies from the laboratories of Drs. Greenberg and Riddell at the Fred Hutchinson Cancer Research Center (FHCRC) have demonstrated that the reconstitution of endogenous CMV-specific T cell responses following allogenic bone marrow transplantation (BMT) correlates with protection from the development of severe CMV disease [58]. In an effort to reconstitute deficient CMV immunity following BMT, CD8+ CMV-specific CTL clones were generated from CMV seropositive HLA-matched sibling donors, expanded, and infused into sibling BMT recipients at risk for developing CMV disease. Fourteen patients were treated with four weekly escalating doses of these CMV-specific CTL clones to a maximum cell dose of 109 cells/m2 without any attendant toxicity [59]. Peripheral blood samples obtained from recipients of adoptively transferred T cell clones were evaluated for in vivo persistence of transferred cells. The recoverable CMV-specific CTL activity increased after each successive infusion of CTL clones, and persisted at least 12 weeks after the last infusion. However, long term persistence of CD8+ clones without a concurrent CD4+ helper response was not observed. No patients developed CMV viremia or disease. These results demonstrate that ex-vivo expanded CMV-specific CTL clones can be safely transferred to BMT recipients and can persist in vivo as functional effector cells that may provide protection from the development of CMV disease.
A complication of bone marrow transplantation, particularly when marrow is depleted of T cells, is the development of EBV-associated lymphoproliferative disease [60]. This rapidly progressive proliferation of EBV-transformed B-cells mimics immunoblastic lymphoma and is a consequence of deficient EBV-specific T cell immunity in individuals harboring latent virus or immunologically naïve individuals receiving a virus inoculum with their marrow graft. Clinical trials conducted at S. Jude's Hospital by Rooney et al. have demonstrated that adoptively transferred ex-vivo expanded donor-derived EBV-specific T cell lines can protect patients at high risk for development of this complication as well as mediate the eradication of clinically evident EBV-transformed B cells [61]. No significant toxicities were observed in the forty-one children treated with cell doses in the range of 4×107 to 1.2×108 cells/m2.
Genetic modification of T cells used in clinical trials has been utilized to mark cells for in vivo tracking and to endow T cells with novel functional properties. Retroviral vectors have been used most extensively for this purpose due to their relatively high transduction efficiency and low in vitro toxicity to T cells [62]. These vectors, however, are time consuming and expensive to prepare as clinical grade material and must be meticulously screened for the absence of replication competent viral mutants [63]. Rooney et al. transduced EBV-reactive T cell lines with the NeoR gene to facilitate assessment of cell persistence in vivo by PCR specific for this marker gene [64]. Riddell et al. have conducted a Phase I trial to augment HIV-specific immunity in HIV seropositive individuals by adoptive transfer using HIV-specific CD8+ CTL clones [65]. These clones were transduced with the retroviral vector tgLS+HyTK which directs the synthesis of a bifunctional fusion protein incorporating hygromycin phosphotransferase and herpes virus thymidine kinase (HSV-TK) permitting in vitro selection with hygromycin and potential in vivo ablation of transferred cells with gancyclovir. Six HIV infected patients were treated with a series of four escalating cell dose infusions without toxicities, with a maximum cell dose of 5×109 cells/m2 [65].
As an alternate to viral gene therapy vectors, Nabel et al. used plasmid DNA encoding an expression cassette for an anti-HIV gene in a Phase I clinical trial. Plasmid DNA was introduced into T cells by particle bombardment with a gene gun [66]. Genetically modified T cells were expanded and infused back into HIV-infected study subjects. Although this study demonstrated the feasibility of using a non-viral genetic modification strategy for primary human T cells, one limitation of this approach is the episomal propagation of the plasmid vector in T cells. Unlike chromosomally integrated transferred DNA, episomal propagation of plasmid DNA carries the risk of loss of transferred genetic material with cell replication and of repetitive random chromosomal integration events.
Chimeric antigen receptors engineered to consist of an extracellular single chain antibody (scFvFc) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (ζ) have the ability, when expressed in T cells, to redirect antigen recognition based on the monoclonal antibody's specificity [67]. The design of scFvFc:ζ receptors with target specificities for tumor cell-surface epitopes is a conceptually attractive strategy to generate antitumor immune effector cells for adoptive therapy as it does not rely on pre-existing anti-tumor immunity. These receptors are “universal” in that they bind antigen in a MHC independent fashion, thus, one receptor construct can be used to treat a population of patients with antigen positive tumors. Several constructs for targeting human tumors have been described in the literature including receptors with specificities for Her2/Neu, CEA, ERRB-2, CD44v6, and epitopes selectively expressed on renal cell carcinoma [68–72]. These epitopes all share the common characteristic of being cell-surface moieties accessible to scFv binding by the chimeric T cell receptor. In vitro studies have demonstrated that both CD4+ and CD8+ T cell effector functions can be triggered via these receptors. Moreover, animal models have demonstrated the capacity of adoptively transferred scFvFc:ζ expressing T cells to eradicate established tumors [73]. The function of primary human T cells expressing tumor-specific scFvFc:ζ receptors have been evaluated in vitro; these cells specifically lyse tumor targets and secrete an array of pro-inflammatory cytokines including IL-2, TNF, IFN-g, and GM-CSF [74]. Phase I pilot adoptive therapy studies are underway utilizing autologous scFvFc:ζ-expressing T cells specific for HIV gp120 in HIV infected individuals and autologous scFcFc:ζ-expression T cells with specificity for TAG-72 expressed on a variety of adenocarcinomas including breast and colorectal adenocarcinoma.
Investigators at City of Hope have engineered a CD20-specific scFvFc:ζ receptor construct for the purpose of targeting CD20+ B-cell malignancy [75]. Preclinical laboratory studies have demonstrated the feasibility of isolating and expanding from healthy individuals and lymphoma patients CD8+ CTL clones that contain a single copy of unrearranged chromosomally integrated vector DNA and express the CD20-specific scFvFc:ζ receptor [76]. To accomplish this, purified linear plasmid DNA containing the chimeric receptor sequence under the transcriptional control of the CMV immediate/early promoter and the NeoR gene under the transcriptional control of the SV40 early promoter was introduced into activated human peripheral blood mononuclear cells by exposure of cells and DNA to a brief electrical current, a procedure called electroporation [77]. Utilizing selection, cloning, and expansion methods currently employed in FDA-approved clinical trials, gene modified CD8+ CTL clones with CD20-specific cytolytic activity have been generated from each of six healthy volunteers in 15 separate electroporation procedures [76]. These clones when co-cultured with a panel of human CD20+ lymphoma cell lines proliferate, specifically lyse target cells, and are stimulated to produce cytokines.
It is desired to develop additional redirected immune cells and, in a preferred embodiment redirected T cells for treating neuroblastoma and other malignancies expressing the CE7 recognized target epitope.